The extraction, concentration and purification of biologically active substances (enzymes, antibodies, hormones, DNA or RNA fragments, and the like) is one of the fundamental problems in biotechnology. The discovery and synthesis of this class of substances, which generally occur with various proportions of other substances present as impurities, has stimulated the search for and development of new techniques and the improvement of known techniques with the object of achieving greater specificity and, where possible, with the greatest economy of time and resources.
These problems of preparative and fractionation techniques, especially as regards the quality (purity) of the products or isolated fractions, take on a special meaning in the case of materials or proteinaceous fractions [sic], commonly composed of various proteins having similar characteristics, often difficult to resolve, as in the case of the fractionation of organ extracts or lysates.
Moreover, this requirement, insofar as it relates to the purity of the biologically active substances and fractions, which is necessary in research work, becomes essential where medicinal products are concerned, as in the case of interferon, insulin, monoclonal antibodies, and the like.
The need to have methods of ever-increasing selectivity has led to the development of other techniques of varying complexity, for example the widely diversified chromatographic techniques such as pawl [sic], high pressure and thin-layer chromatography and, more recently, molecular exclusion chromatography and affinity chromatography, combined in practice with other techniques based on other principles, such as electrophoresis, dialysis, and the like.
Molecular exclusion chromatography is based on the size and/or geometry of the solute or to isolate [sic] or purified, while ion exchange chromatography is based on the coulombic interaction between the substrate and the solute and the acid/base properties of the solute.
For the purposes of the present invention, it is appropriate to stop and consider this chromatographic variant known as "affinity chromatography" (Hay and Dean; Chem. and Ind. 1981; p. 726), based on specific non-covalent interactions between the substance of interest and a suitable reactant immobilized on a suitable substrate (also known as matrix), an interaction which obviously does not take place with the other components (or impurities) present in the sample. In distinction to the other components, the substance of interest remains bound or immobilized on the substrate. This occurs, for example, with certain enzymes (in the sample) which bind strongly but reversibly to specific coenzymes, or an antibody binds to and becomes immobilized with the specific antigen (or vice versa). The next steps are obvious: the combination or complex of substance of interest with the substrate may be processed once separated from the unbound components of the sample, the complex is subsequently split and the substance of interest is recovered, naturally with a higher degree of purity.
This property is exploited for isolating one enzyme from others (or from other solutes) by making use of affinity chromatography, binding the specific coenzyme or the cofactors (AND, AMP) for the enzyme to be separated or purified covalently to a suitable functional group located at the surface of the particles which make up the support of the chromatographic column ("spacer arms"), and then eluting with a solution of the free ligand (coenzyme). The same principle underlies the application of other bonding species such as inhibitors, antigens, antibodies, lectins, and the like.
As a support material, certain polysaccharides such as agarose and dextrans (for example the commercial products known as Sepharose or Sephadex, and the like), or polyacrylates (for example the commercial products known as Bio-Gel, Trisacryl, Ultrogel, and the like), are commonly used. The support material is usually activated, before the operation of binding the chosen ligand, with cyanogen bromide, for example. The largest possible activator/support ratio is generally applied, with the object of achieving a high binding capacity in the chromatographic column.
Recently, the resolving power of affinity chromatography columns has been improved through the application of "spacer arms", designed to compensate for the steric hindrance features resulting from the size and geometry of biologically active molecules of interest. Said spacer arms (aliphatic groups such as, for example, polymethylene chains of 1 to 10 carbon atoms) are pendant groups which project from the surface of the particles of the support, at the free end of which the ligand groups are bound (Low, C. R: Topics in Enzyme and Fermentation Biotechnology, 5a. ed, Wiseman, A: Ellis Horwood, Chichester, 1981, chap. 2).
The resolving power of affinity chromatography has undergone an unexpected improvement as a result of the discovery of a clear affinity between dyes and biologically active molecules, especially proteins, which has led to the fractionation of these biologically active molecules by affinity chromatography, supports and/or colored matrices being employed, as disclosed, for example, in:
Eur. Pat. Sol. EP 183198 (Asahi Chemical Ind. Co. Ltd.) PA0 PCT Int. Sol. WO 84/4309 (Pharmacia AB) PA0 Eur. Pat. Sol. EP 27262 (Dupont de Nemours E.I. and Co.) PA0 USSR SU 1168564 (All Union Scientific Research Inst. of applied Microbiology)
There have been developed, in addition, other methods of this type (affinity chromatography) for the purification of proteins making use of more complicated processes, such as the use of two dyes, one immobilized on the matrix and the other present in the elution fluid (Ger Offen. DE 3244006), an immobilized carrier coupled to a blue dye (Affi-gel Blue) with a residue chelated with a metal (Eur. Pat. Appl. E.P. 94672 A1) or the formation of two phases by addition to the biological fluid of a Cibacron Blue-Sepharose 68 [sic]/PEG 4000 complex, with a suitable buffer (PCT INT. WO 84/4309 A1).
Methods for purifying proteins solely of high molecular weight (British Patent 2053926) are also known.
Thus, the affinity of certain dyes, and of certain matrices with dye bound to them, for substances with biological activity, including proteins, peptides, hormones, enzymes, growth and transforming factors, nucleic acids and nucleotides, has been disclosed in the prior art. This affinity has been applied for the fractionation, purification, and the like, of these substances, employing columns or matrices in which the dye molecules are bound to a colorless original matrix (formed from agarose, dextrans, polyacrylates, and the like, and combinations and variants thereof), either directly or with the interposing of "spacer arms" (Lowe op. cit.). These spacer arms permit greater steric freedom in proximity to the ligand.
In addition, the preparation and application of colored polymers, composed of finely divided or micronized pigments dispersed in a polymer matrix, for industry for purely decorative purposes, in the textile industry, packaging, and the like, covering the whole range of colors of the visible spectrum, have also been disclosed in the prior art; this includes the use of fluorescent dyes, according to a recent disclosure in U.S. Pat. No. 4,016,133 of Ilyoshu et al. An important innovation in the art of preparing colored polymers has been provided by the application of conventional techniques in the polymerization of colorless prepolymers with monomers carrying at least one covalently bound chromophor group. This hence gives rise to permanently colored polymers in which the chromophor group or structure is covalently bound to the hydro-carbon skeleton of the polymer; such is the case with the polymers obtained by Winnik et al. U.S. Pat. No. 4,795,794, which deals with the dispersion polymerization of colorless vinyl monomers and vinyl monomers covalently bound to a dye. As disclosed by Winnik et al., this class of colored polymers are [sic] especially useful for the development of images in electrographic printing processes (colored toner particles).